1. Field of the Invention
The present invention relates to methods and apparatus for detecting amyloid-containing lesions in tissue using autofluorescence. The invention also relates to methods of detecting the onset of cerebral amyloidosis by detecting the presence of amyloid-containing plaques and other related lesions in brain tissue.
2. Description of Related Art
The term xe2x80x9camyloidosisxe2x80x9d encompasses a number of pathological conditions characterized by the deposition of abnormal fibrils (xe2x80x9camyloid fibrilsxe2x80x9d) in extracellular spaces. The amyloid fibril, in turn, represents a final common pathway for a diverse array of proteins. Regardless of their biochemical composition, however, all types of amyloid fibrils share (a) a xcex2-pleated sheet structure, (b) staining similarities, such as green birefringence under polarized light after staining with Congo Red dye, (c) a fibrillar morphology that has a typical electron-microscopic appearance, and (d) other physicochemical properties well known in the art.
The deposition of amyloid fibrils can affect several organs in the systemic forms of the disorder, exemplified by familial Mediterranean fever, familial amyloid polyneuropathy and systemic amyloidosis, or it can be restricted to one organ in localized forms. Among the latter are conditions classified under the rubric xe2x80x9ccerebral amyloidosis,xe2x80x9d which covers the Alzheimer group of diseases, namely, Alzheimer""s disease (pre-senile dementia, senile dementia); Alzheimer""s disease associated with Down""s syndrome; familial Alzheimer""s Disease; genetic Alzheimer""s disease due to mutations such as Presenilin 1, Presenilin 2, and others; Alzheimer""s disease associated with other central-nervous-system diseases, such as Parkinson""s disease, Lewy Body Disease, and cerebrovascular diseases; congophilic angiopathy (associated or not associated with Alzheimer""s disease, familial or not familial), and other disorders and diseases such as those disclosed in U.S. Pat. No. 6,001,331, the disclosure of which is incorporated by reference herein in its entirety.
Alzheimer""s disease (AD) is a complex neurodegenerative disorder characterized by progressive impairments in memory, behavior, language, and visuo-spatial skills, ending ultimately in death. Hallmark pathologies within vulnerable regions include extracellular xcex2-amyloid deposits, intracellular neurofibrillary tangles, synaptic loss, and extensive neuronal cell death. Research on the causes and treatments of Alzheimer""s disease has led investigators down numerous avenues. Although many models have been proposed, no single model of AD satisfactorily accounts for all neuropathologic findings as well as the requirement of aging for disease onset, with the exception of that disclosed in Averback, WO 98/34643, the disclosure of which is incorporated by reference herein in its entirety. Considerable evidence has implicated alterations in production or processing of the human amyloid precursor protein (APP) in the etiology of the disease. However, intensive research has proven that AD is a multifactorial disease with many different, perhaps overlapping, etiologies.
Because of this, those in the field have conducted significant research studies and clinical investigations to study the structural deficiencies, chemical changes, and functional abnormalities both within the brain and within different populations of nerve cells. The depth of such investigations and studies are represented by the following publications, which represent only a handful of the vast reports in this arena: Neurobiology of Alzheimer""s Disease (D. Dawbarn and S. J. Allen, Editors), Bios, Oxford 1995; Dementia, (J. Whitehouse, Ed.), F. A. Davis Company, Philadelphia, 1993; Alzheimer""s Disease: Senile Dementia and Related Disorders (Katzman, R, and R. L. Bick, Eds), Raven Press, New York, 1994, pages 47-51; Alzheimer""s Disease and Related Disorders, Etiology, Pathogenesis and Therapeutics (Iqbol, K., et al., Eds.), Wiley, Chichester, 1999; Alzheimer""s Disease: Advances in Clinical and Basic Research (Corain, B, Ed.), Wiley, New York, 1993; Alzheimer""s Disease: Clinical and Treatment Perspectives (Cutler, N. R., et al., Eds.), Wiley, Chichester, 1995; Alzheimer""s Disease: Therapeutic Strategies (Giacobini, E., Becker, R., Eds.), Birkhauser, Boston, 1994; Paykel, et al., Arch. Gen. Psychiat., 51:325-332 (1994); Amaducci, et al., Neurology, 36:922-931 (1986); McKhann, et al., Neurology 34:939-944 (1984), Heston et al., Arch. Gen. Psychiatry 38:1085-1090 (1981); Aging of the Brain (Gispen and Traber, editors), Elsevier Science Publishers, Amsterdam, 1983, pages 275-282; Heyman et al., Ann. Neurol 15:335-341 (1984); Brayne C. and P. Calloway, Lancet 1:1265-1267 (1988); Roth et al., Br. J. Psychiatry 149:698-709 (1986); Medical Research Council, Report from the NRC Alzheimer""s Disease Workshop, London, England, 1987; Morris et al., Neurology 41:469-478 (1991); and the references cited within each of these publications.
To date, Alzheimer""s disease is the third most expensive disease in the United States, costing society approximately $100 billion each year. It is one of the most prevalent illnesses in the elderly population, and with the aging of society, will become even more significant. Costs associated with AD include direct medical costs such as nursing home care, direct nonmedical costs such as in-home day care, and indirect costs such as lost patient and care giver productivity. Medical treatment and behavior modification may have economic benefits by slowing the rate of cognitive decline, delaying institutionalization, reducing care giver hours, and improving quality of life. Pharmacoeconomic evaluations have shown positive results regarding the effect of drug therapy and behavior modification on nursing home placement, cognition, and care giver time.
Despite the array of research investigations and studies undertaken to date, present clinical evaluations still have a difficult time establishing an unequivocal diagnosis of Alzheimer""s Disease. Autopsy or biopsy is widely considered the gold standard method for AD diagnosis. Different criteria exists that assess and determine the presence of neurofibrillary tangles (NFT), cell loss and senile (amyloid) plaques in brain tissue. These criteria for the definite diagnosis of Alzheimer""s Disease are met only by histologic evidence.
The research to date has been diverse insofar as the causes of various forms of cerebral amyloidosis. That is, the research has varied with respect to how the actual amyloid plaques and other similar lesions form in the brain. There is little disagreement in the scientific community, however, that a universally accepted indicator of cerebral amyloidosis is the accumulation of large numbers of amyloid-containing lesions, so-called xe2x80x9csenile plaques,xe2x80x9d that are comprised in large part of amyloid fibrils. Senile plaques are spherical, ranging from 10 to 200 xcexcm in diameter, and are found occasionally in aged adult cerebral cortex, but are found in large numbers in Alzheimer-affected cerebral cortex. To date, the best means by which one can measure the presence of senile plaques in the brain is achieved by a brain biopsy or a postmortem examination, and subsequent detection of amyloid plaques.
In this context, various mechanisms have been described by which one can detect amyloid plaques and other amyloid-related lesions. Amyloid plaques and amyloid lesions can be visualized in histological sections by staining with many methods, such as silver impregnation, eosin, periodic acid Schiff, Congo red, thioflavins, and others. Amyloid plaques and amyloid lesions also can be visualized by immunohistochemical methods of staining the amyloid and other proteins in the plaques by antibodies to plaque proteins conjugated to enzymes, such as alkaline phosphatase or horseradish peroxidase and others, or to fluorophores such as fluorescein isothiocyanate, rhodamine, or others.
For example, Dowson reported detection of senile plaques in a post mortem thin slice of brain tissue by fluorescence microscopy. Dowson, J. H., Histopathology, 5:305-310 (1981). The thin slices were epi-illuminated with ultraviolet light having a wavelength between 340-380 nm, and the excited light was detected at wavelengths above 430 nm, with the best results around 460 nm. Other means of detecting amyloid plaques have been achieved by staining the thin sections of tissue with a fluorochrome, irradiating the tissue with various wavelengths of light, and detecting the emitted light. Congo Red often is used to stain the thin-section tissue samples to detect the presence of optically anisotropic materials that display congophilic birefringence. This optical phenomenon is recognized as being caused by amyloid plaques and other amyloid-containing components, such as dense microspheres (DMS, or xe2x80x9cspheronsxe2x80x9d). See, U.S. Pat. No. 5,567,720, the disclosure of which is incorporated by reference herein in its entirety.
Methods of staining thin tissue sections are not the same as methods for solving the problems of detecting a signal in whole tissue, and there can be no assurances that a method used on a thin microscopic section can be applied to whole tissue. For example, exogenous chromophores or fluorophores or radiocontrast media, or conjugated fluorophores or chromophores, in vivo usually need to be administered by circulation through the bloodstream, by ingestion by the gastrointestinal route, or by inhalation, etc. Trying to identify particular fluorescence materials in a mass of tissue is complicated further by the number of different tissue fluorophores such as lipofuscin, which autofluoresces yellow and green in a wide range of 400 to 600 nm to excite and 400-640 to detect, and which is ubiquitous in AD brain. Other difficulties encountered in detecting a signal in whole tissue are described in more detail below.
Scientists have attempted to separate amyloid plaque material from postmortem brain by use of fluorescence-activated cell sorting (FACS). Selkoe, et al., have reported a method of purifying amyloid senile plaques from brain tissue by sorting the core plaques using FACS. J. Neurochem., Vol. 46, No. 6, 1820-1834 (1986); and Methods in Enzymology, Vol. 134, No. 37, 388-405 (1986). The amyloid plaque cores were detected and separated from the surrounding tissue by using excitation light at 488 nm and emission recognition at 580 nm.
Hanlon reports the ability to distinguish AD brain tissue from non-AD brain tissue using autofluorescence at near infrared wavelengths. Hanlon, et al., Photochemistry and Photobiology, 70(2): 236-242 (1999). Hanlon describes exciting tissue samples of AD brain tissue and non-AD brain tissue by irradiating the samples with light at about 647 nm. The samples then are detected with a detection wavelength in the range of from about 665-850 nm. Hanlon reports the ability to distinguish AD brain tissue from non-AD brain tissue using this method, but does not recognize that any signal emanates from amyloid plaques, or that the signal is generated by an amyloid-containing deposit.
All of this research has led down many different paths to the detection of amyloid plaques and other amyloid-related components in thin sections of post mortem brain. There has yet to be developed a relatively non-invasive endogenous method that can be employed without administration of exogenous chromophore of fluorophore by which the specific presence of amyloid plaques, amyloid-containing deposits, and related lesions can be detected in a living animal. This is not surprising given the variety of methods known to detect the presence of amyloid-containing materials in postmortem brain. The various staining techniques with chromophores and fluorophores cannot thus far be used on a living animal, and the autofluorescence techniques to date have suggested a vast array of wavelengths used to test the tissue; Dowson suggesting 340-380 nm for thin-sections of post-mortem brain tissue and 460 nm to detect, Selkoe suggesting 488 nm for FACS of homogenized post-mortem brain tissue, Christie suggesting 720 nm, and Hanlon suggesting the use of near infrared light at 647 nm to excite and 665-850 nm to detect. Trying to identify particular fluorescence materials in a mass of tissue is complicated further by the plethora of divergent tissue fluorophores such as lipofuscin, which autofluoresces yellow and green in a wide range of 400 to 600 nm to excite and 400-640 to detect, and which is ubiquitous in AD brain.
Indeed, the nature of the human brain tissue is such that the use of autofluorescence to detect cell abnormalities produces a large amount of data, most of which is extraneous to the intended measurement. A primary reason for this situation is that tissues contain an extensive and diverse assortment of fluorescent species. Many of the species are present in high concentrations and have excitation bands distributed throughout the ultraviolet and the visible spectra regions.
Another limitation is that the emission band of one fluorophore may overlap the excitation band of another fluorophore, consequently leading to energy transfer between the emission and excitation bands. Consequently, emissions from one fluorophore could possibly excite another fluorophore. The net effect is that exciting a tissue sample at almost any wavelength in the ultraviolet, visible, or infrared wavelength regions causes tissue autofluorescence over a broad spectral range.
It is even more difficult to detect the presence of autofluorescent tissue in the brain because most wavelengths of light will not pass through the skull, and those that do, may cause damage to the tissue. Hanlon discussed above suggests a non-invasive method of diagnosing Alzheimer""s Disease using near infrared fluorescent spectroscopy. Backsai, et al., and Skovronsky, et al., disclose imaging techniques such as staining, fluorescent labeling, or radiolabeled ligands that recognize senile plaques that purport to be useful in detecting amyloid plaques in vivo. Injecting or otherwise inserting these compounds into the brain can be problematic given the toxicity and blood-brain barrier concerns.
Recent developments in endoscope technology and fiber optics have led to the production of small and effective imaging and detecting apparatus useful in autofluorescence. For example, U.S. Pat. Nos. 6,147,800, 6,091,984, 6,081,740, 5,507,287, 5,590,660, 5,999,844, and 4,930,516 disclose various apparatus and methods for imaging diseased, often cancerous tissue using autofluorescence. The disclosure of each of these patents is incorporated by reference herein in its entirety. Other autofluorescence apparatus are known in the art (e.g., Pentax SAFE-1000 Autofluorescence-System, available from Pentax-endoscopy.)
The art therefore proposes a number of different approaches to detecting the presence of amyloid plaques and other amyloid-containing lesions, none of which has been proven successful in this regard. While there has yet to be a cure for Alzheimer""s Disease, there is effective temporary symptomatic treatment. Diagnosis of AD would be beneficial so that those inflicted therewith could obtain timely treatment to minimize its effects. Diagnosis also is critical for many other reasons such as planning for care, for reducing anxiety when diagnosis is negative, for investigation of putative treatments, and for monitoring therapies.
There exists a need to develop a method of detecting amyloidosis in the brain and other organs to diagnose conditions involving amyloid-containing deposits and other related lesions such as Alzheimer""s disease and other related conditions. There also exists a need to develop a method of detecting amyloidosis in a living animal, preferably without the administration of exogenous chemicals such as fluorophores, chromophores, or antibodies conjugated to fluorphores, chromophores, etc. In addition, there exists a need to develop a system that can detect amyloidosis in the brain and other organs to diagnose conditions involving amyloid-containing deposits.
It is therefore a feature of an embodiment of the invention to provide a method of detecting amyloidosis in the brain and other organs. The method is useful in diagnosing conditions involving amyloid-containing deposits such as Alzheimer""s disease and related conditions. It is an additional feature of an embodiment of the invention to provide a system for detecting amyloidosis to diagnose conditions involving amyloid-containing deposits. These and other features of the invention will be readily apparent to those skilled in the art upon reading the description herein.
In accordance with these and other features of the invention, there is provided a method of detecting amyloidosis in a mammal that includes subjecting tissue of a mammal suspected of having at least one form of amyloidosis to autofluorescence-initiation, and detecting the presence of autofluorescence-emitted light having a wavelength of from about 400 nm to about 460 nm.
In accordance with another feature of the invention, there is provided a method of detecting amyloidosis in a mammal that includes subjecting tissue of a mammal suspected of having at least one form of amyloidosis to excitation incident radiation from a light source having a wavelength of light within the range of from about 360 to about 370 nm, and to illumination incident radiation from a light source having wavelengths in the visible spectrum, to produce an emitted light signal beam and a reflected light signal beam, separating the emitted light signal beam from the reflected light signal beam, and detecting the presence of emitted light having a wavelength of from about 400 nm to about 460 nm.
In accordance with an additional feature of an embodiment of the invention, there is provided a method of detecting amyloidosis in a mammal that includes subjecting circulating fluid of a mammal suspected of having at least one form of amyloidosis to excitation incident radiation from a light source having a wavelength of light within the range of from about 360 to about 370 mn, and to illumination incident radiation from a light source having wavelengths in the visible spectrum, to produce an emitted light signal beam and a reflected light signal beam, separating the emitted light signal beam from the reflected light signal beam, and detecting the presence of emitted light having a wavelength of from about 400 nm to about 460 nm.
In accordance with another feature of an embodiment of the invention, there is provided a system for detecting amyloidosis in a mammal that includes a radiation source that illuminates tissue or circulating fluid of a mammal suspected of having at least one form of amyloidosis with: (i) excitation incident radiation having a wavelength of light within the range of from about 360 to about 370 nm; and (ii) illumination incident radiation having wavelengths in the visible spectrum. The system also includes a mechanism for collecting an emitted light signal beam from the tissue or circulating fluid, and a reflected light signal beam from the tissue or circulating fluid, and then separating the emitted light signal beam from the reflected light signal beam. The system further includes a mechanism that is capable of detecting the presence of emitted light having a wavelength of from about 400 nm to about 460 nm.
These and other features of the invention will be readily apparent to those skilled in the art upon reading the detailed description that follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description. Unless otherwise specified, the respective contents of the documents cited herein are hereby incorporated by reference in their entirety.